Stoichiometry and Architecture of the Vesicle Fusion Machine in PC-12 Cells

PC-12 细胞中囊泡融合机的化学计量和结构

• 批准号: 7530937
• 负责人: James C. Weisshaar
• 金额: $ 18.33万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7530937
• 关键词: Alzheimer' s Disease; Architecture; Axon; Binding; Blood Circulation; Caliber; Cell membrane; Cells; Cellular Membrane; Chemicals; Chimeric Proteins; Chromosome Pairing; Color; Communication; Complex; Count; Dendrites; Docking; Endocrine; Event; Exocytosis; Extracellular Space; Fluorescence; Fluorescence Microscopy; Freezing; Funding Mechanisms; Goals; Growth Factor; Hippocampus (Brain); Image; In Vitro; Individual; Interphase Cell; Label; Length; Localized; Location; Measurement; Measures; Methodology; Microscope; Microscopy; Molecular; Neurodegenerative Disorders; Neurons; Neuropeptides; Neurotransmitters; Numbers; Optics; PC12 Cells; Parkinson Disease; Photobleaching; Positioning Attribute; Process; Proteins; Public Health; Recycling; Relative (related person); Resolution; Running; SNAP receptor; Sampling; Secretory Vesicles; Side; Signal Transduction; Site; Standards of Weights and Measures; Structure; Study models; Synapses; Synaptic Cleft; Synaptic Vesicles; Techniques; Testing; Transfection; Travel; Use of New Techniques; Vesicle; Water; Width; Work; base; cell fixing; controlled release; design; fluorescence microscope; fluorophore; insight; interest; photoactivation; receptor; research study; sample fixation; sensor; size; spatial relationship; stoichiometry; synaptotagmin; synaptotagmin I; syntaxin; syntaxin 1; vesicle-associated membrane protein; voltage

DESCRIPTION (provided by applicant): Neurons communicate with each other by transmitting chemical signals from cell to cell. In the key process of neuronal exocytosis, a voltage wave travels the length of the transmitting cell to open Ca2+ channels in the plasma membrane (PM) at the axon terminus. The abrupt influx of Ca2+ triggers exocytosis, the process by which secretory vesicles docked at the PM open a fusion pore and spill their neurotransmitter into the synaptic cleft. The neurotransmitter is taken up by receptors placed on the opposite side of the synapse in the PM of the receiving neuron's dendrite. This in turn stimulates a second voltage wave that travels the length of the receiving neuron. The same process of exocytosis enables endocrine cells to emit triggered chemical signals into the intercellular space or the bloodstream. The vesicle fusion machine is the complex of proteins that cause exocytosis. Key components include the SNARE (soluble NSF attachment receptor) proteins syntaxin (Syx), SNAP-25, and synaptobrevin (Syb), and the Ca2+ sensor synaptotagmin (Syt). Essentially nothing is known about the stoichiometry (number of working copies of each protein) or the overall architecture (spatial relationships among the proteins) of the fusion machine, either before or after the Ca2+ influx. The goal of this work is to use the new fluorescence technique of photoactivated localization microscopy (PALM) to count and to locate fusion machine proteins with ~5-nm accuracy in fixed PC-12 cells, a standard model for the study of exocytosis. PALM uses a long sequence of activation-location-photobleaching cycles to locate all labeled copies of a protein one by one. In each experiment, the photoactivatable fluorescent protein mEos will be genetically attached to a particular fusion protein (Syx, SNAP-25, Syb, or Syt). For each cell studied, the LDCVs (large, dense-core secretory vesicles) will also be located to ~5 nm by co-expression of the neuropeptide eGFP-ANF, which trafficks to LDCVs and exocytoses normally. Comparison of PALM images of resting cells vs depolarized cells may reveal structural changes induced by the Ca2+ influx. New structural information is critically important to progress in understanding the underlying molecular mechanism of triggered vesicle fusion. PALM imaging has the potential to reveal the structure of the fusion machine before and after Ca2+ influx at an entirely new level of detail. Existing hypotheses about rings of Syx/SNAP-25 centered beneath a docked vesicle and about binding relationships between Syt and Syx, among many others, will be tested directly. In the longer term, extensions to PC-12 cells differentiated by culture in neuron growth factor (NGF) and to hippocampal neurons are envisioned. The new methodology developed here should find widespread application in structural studies of other large protein complexes embedded in cellular membranes. PUBLIC HEALTH RELEVANCE: The goal of this work is to use a new high-resolution fluorescence microscopy technique to determine the number and arrangement in space of key proteins in the vesicle fusion machine. That machine is the complex of proteins that controls the release of neurotransmitters into the synapse, the key step in neuron-neuron communication. In the long run, a fundamental understanding of how neurons work at the molecular level should help elucidate the underlying causes of neurodegenerative diseases such as Parkinson's and Alzheimer's disease.

描述（由申请人提供）：神经元通过在细胞间传递化学信号相互通信。在神经元胞吐的关键过程中，电压波沿传递细胞的长度行进，以打开轴突末端质膜（PM）中的Ca 2+通道。Ca2+的突然流入触发胞吐作用，通过该过程，停靠在PM的分泌囊泡打开融合孔并将其神经递质溢出到突触间隙中。神经递质被置于接受神经元树突的PM中突触对面的受体吸收。这反过来又刺激了第二个电压波，该电压波沿着接收神经元的长度传播。同样的胞吐过程使内分泌细胞能够将触发的化学信号发射到细胞间隙或血流中。囊泡融合器是引起胞吐作用的蛋白质复合物。关键成分包括SNARE（可溶性NSF附着受体）蛋白质突触融合蛋白（Syx）、SNAP-25和小突触蛋白（Syb）以及Ca 2+传感器突触结合蛋白（Syt）。基本上，在Ca2+流入之前或之后，对融合机器的化学计量（每个蛋白质的工作拷贝数）或整体结构（蛋白质之间的空间关系）一无所知。这项工作的目标是使用新的荧光技术的光激活定位显微镜（PALM）计数和定位融合机器蛋白在固定的PC-12细胞，一个标准模型的研究胞吐作用的精度为5 nm。PALM使用一个长序列的激活-定位-光漂白循环来逐个定位蛋白质的所有标记拷贝。在每个实验中，可光活化的荧光蛋白mEos将被遗传地连接到特定的融合蛋白（Syx、SNAP-25、Syb或Syt）。对于所研究的每个细胞，LDCV（大的致密核心分泌囊泡）也将通过神经肽eGFP-ANF的共表达定位到~5nm，其运输到LDCV并正常地胞吐。静息细胞与去极化细胞的PALM图像的比较可以揭示由Ca2+内流引起的结构变化。新的结构信息对于理解触发囊泡融合的潜在分子机制至关重要。PALM成像有可能以全新的细节水平揭示Ca2+流入前后融合机的结构。现有的假设环Syx/SNAP-25中心下停靠囊泡和Syt和Syx之间的结合关系，以及许多其他的，将直接进行测试。从长远来看，扩展到PC-12细胞分化的神经元生长因子（NGF）和海马神经元的文化是可以预见的。在这里开发的新方法应该找到广泛的应用在其他大型蛋白质复合物嵌入在细胞膜的结构研究。公共卫生关系：这项工作的目标是使用一种新的高分辨率荧光显微镜技术来确定囊泡融合机中关键蛋白质的数量和空间排列。这台机器是一种蛋白质复合体，它控制着神经递质释放到突触中，这是神经元之间通信的关键步骤。从长远来看，对神经元如何在分子水平上工作的基本理解应该有助于阐明神经退行性疾病（如帕金森病和阿尔茨海默病）的根本原因。